The project described here attempts to further characterize detergent-insoluble glycosphingolipid enriched (DIG) fractions from rat Sertoli cells. These fractions are enriched in membrane microdomains (rafts), located at the apical surface of the Sertoli cell where interactions with the meiotic and post-meiotic spermatogenic cell occur. This project focuses on three major areas of investigation. First, based on the significant enrichment of Ras in these DIG fractions, and no apparent enrichment of Raf, experiments are proposed to immunoprecipitate DIG fractions using anti-Ras antibody and look for co-precipitating proteins using SDS-PAGE, blotting and immunostaining. Of particular interest is phosphatidylinositol 3-kinase (PI3K), as well as downstream enzymes such as phosphoinositide-dependent kinase (PDK1), protein kinase B (PKB), protein kinase C (PKC) and phospholipase C (PLC). Experiments with spermatogenic cell conditioned media will also be conducted along with SDS-PAGE, blotting, and immunostaining to determine what, if any, proteins segregate into DIG fractions, or are selectively tyrosine phosphorylated, following exposure to the spermatogenic cell-derived signals. Secondly, we propose to re-examine iron transport, both influx and efflux, in the presence and absence of the GPI-anchored ceruloplasmin that has been demonstrated to be a major protein in Sertoli cell DIG fractions. For examination of 55Fe efflux, Sertoli cells will be pre-loaded, and for 55Fe influx, the iron will be added to the apical chamber of the bicameral culture dish. Influx and efflux will then be measured in the presence and absence of ceruloplasmin. Removal of ceruloplasmin from the cell surface will be done with the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC). Finally, experiments will be done to identify the dynamin isoform(s) that has been observed to segregate into the Sertoli cell DIG fractions. Note that dynamin with its pleckstrin homology domain sorts into raft-like domains and also functions in endocytosis/exocytosis in many cells. Immunoprecipitation experiments will also be done to identify interacting proteins. Using immunohistochemistry, distribution studies of the dynamin isoforms in whole testis sections will be done. In summary, these experiments are all focused on defining the role of these Sertoli cell rafts in Sertoli-spermatogenic cell interactions. The very difficult task of defining the major events of Sertoli-spermatogenic cell interactions, so essential for successful spermatogenesis, may be made easier by the ability to isolate these membrane microdomains and identify the players involved.